Method of enhancing delivery of therapeutic compounds to the eye

ABSTRACT

The invention provides methods for enhancing the delivery of therapeutic compounds to the eye of a subject by administering plasmin or derivatives thereof and the therapeutic compounds to the eye.

RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/777,420 filed on Sep. 15, 2015, which is a 371 of InternationalApplication No. PCT/US2014/026224, filed Mar. 13, 2014, which claimspriority to, and the benefit of, U.S. Provisional Application No.61/785,015, filed on Mar. 14, 2013; the contents of each of which arehereby incorporated by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under EY017130 awardedby the National Institutes of Health. The government has certain rightsin the invention.

INCORPORATION OF SEQUENCE LISTING

The contents of the text file named “RTRO-704/C01US_SeqList.txt,” whichwas created on Aug. 14, 2017 and is 25.7 KB in size, are herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates generally to methods of enhancing the delivery oftherapeutic compounds to the eye.

BACKGROUND OF THE INVENTION

The eye is a complex optical system that detects light, converts thelight to a set of electrical signals, and transmits the signals to thebrain, ultimately generating a representation of our world. Oculardiseases and disorders can cause diminished visual acuity, diminishedlight sensitivity, and blindness.

Delivery of therapeutic compounds to specific ocular tissues affected byan ocular disease or disorder, such as the retina, is a challenge.Current methods, such as intravitreal injection or implanted drugdelivery devices, are still limited in the efficacy of delivery.Specifically, the therapeutic agents are often localized only to theimmediate areas surrounding the delivery site, and fail to permeate ordiffuse beyond intervening ocular structures or throughout the targetedocular tissue, thereby severely limiting the efficacy of suchtherapeutics. Thus, there exists a long-felt need for methods to enhancethe delivery of therapeutic compounds to the eye.

SUMMARY OF THE INVENTION

The invention provides a solution for the long-felt need for methods toenhance or improve the delivery of therapeutic compounds to the eye.

The present invention features a method of enhancing the delivery of atherapeutic agent to an eye of a subject by administering a plasmin orderivative thereof and the therapeutic agent to the eye. The presentinvention also features the use of a composition comprising a plasmin orderivative thereof for delivery to the eye of a subject for enhancingthe delivery of a therapeutic agent.

In one aspect, the plasmin or derivative thereof is a miniplasmin or amicroplasmin (Ocriplasmin). The plasmin or derivative thereofencompassed in the present invention includes amino acid sequences SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or functional variants or fragmentsthereof.

In one aspect, the therapeutic agent is selected from a small molecule,a nucleic acid, an antibody, or a peptide. The nucleic acid is a nucleicacid expression vector (i.e., a viral vector), a plasmid, or an siRNA.For example, the viral vector is a AAV viral vector (i.e., recombinantAAV or rAAV) that encodes a transgene. Preferably, the transgene encodesa gene product that increases or restores light sensitivity, increaseslight detection, increases photosensitivity, increases visual evokedpotential, or restores vision to the blind. More preferably, thetransgene is an opsin gene. Examples of opsin genes include, but are notlimited to, channelrhodopsins (i.e., channelrhodopsin-1,channelrhodopsin-2, Volvox carteri channelrhodopsins 1 or 2),melanopsin, pineal opsin, photopsins, halorhodopsin, bacteriorhodopisin,proteorhodopsin, or any functional variants or fragments thereof.

Other examples of therapeutic agents include, but are not limited toranibizumab antibody FAB (Lucentis), VEGF Trap fusion molecule (VEGFTrap-Eye), macugen pegylated polypeptide (Pegaptanib), and bevacimzumab(Avastin). Any of the therapeutic agents used in the present inventionmay be encapsulated in a nanoparticle, a polymer, or a liposome.

In one aspect, the plasmin or derivative thereof and the therapeuticagent are delivered concurrently or sequentially.

The present invention provides a method in which the therapeutic agentis delivered to a retinal cell. The retinal cell is a retinal ganglioncell, a retinal horizontal cell, a retinal bipolar cell, an amacrinecell, a photoreceptor cell, a Müller glial cell, or a retinal pigmentepithelial cell.

In one aspect, the plasmin or derivative thereof and the therapeuticagent is administered to the vitreous of the eye.

The present invention further provides a method of increasing orrestoring light sensitivity in a subject comprising administering aplasmin or derivative thereof and a viral vector that encodes an opsinto the vitreous of the eye. The present invention also provides a methodof improving or restoring vision in a subject comprising administering aplasmin or derivative thereof and a viral vector that encodes an opsinto the vitreous of the eye.

Uses of a composition comprising a plasmin or derivative thereof fortreating an ocular disease or disorder in a subject are also providedherein.

The subject is suffering from an ocular disease or disorder. Preferably,the ocular disease or disorder is associated with photoreceptordegeneration.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the present invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are expressly incorporated byreference in their entirety. In cases of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples described herein are illustrative onlyand are not intended to be limiting.

Other features and advantages of the invention will be apparent from andare encompassed by the following detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a series of representative GFP fluorescence images in retinalvertical sections after intravitreal injection of AAV2 vectors (6×10¹²vg/ml), AAV2/2-ChR2-GFP-WPRE-hGHpA, in control (A, B) or co-injectionwith plasmin (0.025 IU/eye) (C, D). The vectors were co-injected alongwith plasmin into the vitreous space of adult C56BL/6J mice at age ofapproximately one month. Transduction efficiency was evaluated one monthafter virus injection by immunostaining and cell counting.

FIG. 2 is a series of representative GFP fluorescence and DAPI stainingimages demonstrating the effects of plasmin on AAV-mediated transductionefficiency and the potential neurotoxicity in retinal ganglion cells.AAV2 vectors (2×10¹² vg/ml), AAV2/2-ChR2-GFP-WPRE-hGHpA, was injected incontrol (A, E), or was co-injected at (B, F) low (L: 0.005 IU), (C, G)middle (M: 0.025 IU), and (D, H) high (H: 0.100 IU plasmin/eye)concentrations. Retinal ganglion cells were stained by DAPI (A-D).

FIG. 3 is two graphs showing the quantitative assessment of the effectsof plasmin on (A) AAV-mediated transduction efficiency and (B) potentialneurotoxicity of plasmin in retinal ganglion cells. Co-injection ofplasmin at low (L: 0.005 IU), middle (M: 0.025 IU), and high (H: 0.100IU plasmin/eye) concentrations significantly increased the AAV-mediatedtransduction efficiency in retinal ganglion cells (A). Co-injection ofplasmin did not show any significant neurotoxicity to retinal ganglioncells. The ganglion cell counts were assessed from multiple unit areasof 223 μm×167 μm. * p<0.05; ** p<0.005.

FIG. 4 is a series of representative fluorescence images ofmCherry-expressing retinal bipolar cells in retinal whole-mounts. AAV2vectors (2×10¹² vg/ml) with Y444F capsid mutation carrying mCherry undercontrol of a mGluR6 promoter were co-injected along with plasmin ofthree doses (L: 0.005 IU, M: 0.025 IU, and H: 0.100 IU/eye) into thevitreous space of adult C56BL/6J mice at age of approximately one month.Transduction efficiency was evaluated one month after virus injection byimmunostaining and cell counting.

FIG. 5 is three graphs showing quantitative data for the effects ofplasmin on AAV-mediated transduction efficiency in retinal bipolarcells. A) Center; B) Mid-region; and C) Periphery. The counts of mCherryexpressing retinal bipolar cell were assessed from multiple unit areasof 223 μm×167 μm. * p<0.05; ** p<0.01; ***p<0.001.

DETAILED DESCRIPTION

The present invention provides methods for enhanced delivery oftherapeutic compounds or agents to the eye of a subject by administeringa plasmin or derivative thereof and the therapeutic agent to the eye. Insome embodiments, the plasmin or derivative thereof and the therapeuticagent may be delivered to the vitreous for enhanced delivery to theretina and retinal cells. The retinal cells include, for example,photoreceptor cells (e.g., rods, cones, and photosensitive retinalganglion cells), horizontal cells, retinal bipolar cells, amacrinecells, retinal ganglion cells, Müller glial cells, and retinal pigmentepithelial cells. In other embodiments, the plasmin or derivativethereof and the therapeutic agent may be delivered to, for example, theposterior segment, the anterior segment, the sclera, the choroid, theconjunctiva, the iris, the lens, or the cornea.

The retina is a complex tissue in the back of the eye that containsspecialized photoreceptor cells called rods and cones. Thephotoreceptors connect to a network of nerve cells for the localprocessing of visual information. This information is sent to the brainfor decoding into a visual image. The retina is susceptible to a varietyof diseases, including age-related macular degeneration (AMD), diabeticretinopathy (DR), retinitis pigmentosa (RP), glaucoma, and otherinherited retinal degenerations, uveitis, retinal detachment, and eyecancers (ocular melanoma and retinoblastoma). Each of these can lead tovisual loss or complete blindness.

Delivery of therapeutic compounds to the retina is a challenge, due tothe complex structure of the eye. Intravitreal injection and vitrealdelivery devices are frequently used to deliver therapeutic compounds tothe retina, however the efficiency of delivery is impaired by the innerlimiting membrane (ILM) and the multiple layers of cells of the retina.

Plasmin

Plasmin is a serine protease that is present in the blood that degradesfibrin blood clots and other blood plasma proteins. Specifically,plasmin cleaves fibrin, fibronectin, thrombospondin, laminin,proaccelerin, and Von Willebrand Factor (VWF) into soluble products.Plasmin exhibits preferential cleavage at the carboxyl side of Lysineand Arginine residues with higher selectivity than trypsin.

Specifically, plasmin originates from a zymogen, or inactive precursorprotein, called plasminogen (PLG). The amino acid sequence ofplasminogen is known in the art, for example, Genbank Accession NumberNP_000292, and listed below:

(SEQ ID NO: 1) MEHKEVVLLLLLFLKSGQGEPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYGNVAVTVSGHTCQHWSAQTPHTHANRTPENFPCKNLDENYCRNPDGKRPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN

The nucleic acid of plasminogen is known in the art, for example,Genbank Accession Number NM_000301, and as listed below:

(SEQ ID NO: 2) GAATCATTAACTTAATTTGACTATCTGGTTTGTGGATGCGTTTACTCTCATGTAAGTCAACAACATCCTGGGATTGGGACCCACTTTCTGGGCACTGCTGGCCAGTCCCAAAATGGAACATAAGGAAGTGGTTCTTCTACTTCTTTTATTTCTGAAATCAGGTCAAGGAGAGCCTCTGGATGACTATGTGAATACCCAGGGGGCTTCACTGTTCAGTGTCACTAAGAAGCAGCTGGGAGCAGGAAGTATAGAAGAATGTGCAGCAAAATGTGAGGAGGACGAAGAATTCACCTGCAGGGCATTCCAATATCACAGTAAAGAGCAACAATGTGTGATAATGGCTGAAAACAGGAAGTCCTCCATAATCATTAGGATGAGAGATGTAGTTTTATTTGAAAAGAAAGTGTATCTCTCAGAGTGCAAGACTGGGAATGGAAAGAACTACAGAGGGACGATGTCCAAAACAAAAAATGGCATCACCTGTCAAAAATGGAGTTCCACTTCTCCCCACAGACCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAACTACTGCAGGAATCCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTACTGATCCAGAAAAGAGATATGACTACTGCGACATTCTTGAGTGTGAAGAGGAATGTATGCATTGCAGTGGAGAAAACTATGACGGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAGGCCTGGGACTCTCAGAGCCCACACGCTCATGGATACATTCCTTCCAAATTTCCAAACAAGAACCTGAAGAAGAATTACTGTCGTAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCCAACAAGCGCTGGGAACTTTGTGACATCCCCCGCTGCACAACACCTCCACCATCTTCTGGTCCCACCTACCAGTGTCTGAAGGGAACAGGTGAAAACTATCGCGGGAATGTGGCTGTTACCGTGTCCGGGCACACCTGTCAGCACTGGAGTGCACAGACCCCTCACACACATAACAGGACACCAGAAAACTTCCCCTGCAAAAATTTGGATGAAAACTACTGCCGCAATCCTGACGGAAAAAGGGCCCCATGGTGCCATACAACCAACAGCCAAGTGCGGTGGGAGTACTGTAAGATACCGTCCTGTGACTCCTCCCCAGTATCCACGGAACAATTGGCTCCCACAGCACCACCTGAGCTAACCCCTGTGGTCCAGGACTGCTACCATGGTGATGGACAGAGCTACCGAGGCACATCCTCCACCACCACCACAGGAAAGAAGTGTCAGTCTTGGTCATCTATGACACCACACCGGCACCAGAAGACCCCAGAAAACTACCCAAATGCTGGCCTGACAATGAACTACTGCAGGAATCCAGATGCCGATAAAGGCCCCTGGTGTTTTACCACAGACCCCAGCGTCAGGTGGGAGTACTGCAACCTGAAAAAATGCTCAGGAACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTAGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACCCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTATTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTAATTGGACGGGAGACAGAGTGACGCACTGACTCACCTAGAGGCTGGAACGTGGGTAGGGATTTAGCATGCTGGAAATAACTGGCAGTAATCAAACGAAGACACTGTCCCCAGCTACCAGCTACGCCAAACCTCGGCATTTTTTGTGTTATTTTCTGACTGCTGGATTCTGTAGTAAGGTGACATAGCTATGACATTTGTTAAAAATAAACTCTGTACTTAACTTTGATTTGAGTAAATTTTGGTTTTGGTCTTCAACATTTTCATGCTCTTTGTTCACCCCACCAATTTTTAAATGGGCAGATGGGGGGATTTAGCTGCTTTTGATAAGGAACAGCTGCACAAAGGACTGAGCAGGCTGCAAGGTCACAGAGGGGAGAGCCAAGAAGTTGTCCACGCATTTACCTCATCAGCTAAGCGAGGGCTTGACATGCATTTTTACTGTCTTTATTCCTGACACTGAGATGAATTTTTCAAAGCTGCAACATGTATGGGGAGTCATGCAAACCGATTCTGTTATTGGGAATGAAATCTGTCACCGACTGCTTGACTTGAGCCCAGGGGACACGGAAGCAGAGAGCTGTATATGATGGAGTGAACCGGTCCATGGTGTGTAACACAAGACCAACTGAGAGTCTGAATGTTATTCTGGGGCACACGTGAGTCTAGGATTGGTGCCAAGAGCATGTAAATGAACAACAAGCAAATATTGAAGGTGGACCACTTATTTCCCATTGCTAATTGCCTGCCCGGTTTTGAAACAGTCTGCAGTACAACACGGTCACAGGAGAATGACCTGTGGGAGAGATACATGTTTAGAGGAAGAGAAAGGACAAAGGCACACGTTTTACCATTTAAAATATTGTTACCAAACAAAAATATCCATTCAAAATACAATTTAACAATGCAACAGTCATCTTACAGCAGCAGAAATGCAGAGAAAAGCAAAACTGCAAGTGATGTGAATAAAGGGTGAAT GTAGTCTCAAATCCTCAAA

The signal peptide sequence of plasminogen is 19 amino acids long. Thus,the plasminogen sequence without the signal peptide encompasses aminoacids from positions 20-810 of the plasminogen sequence. The signalpeptide sequence is as follows:

(SEQ ID NO: 3) MEHKEVVLLLLLFLKSGQG

The plasmin heavy chain A is 561 amino acids, comprising the amino acidsequence provided below:

(SEQ ID NO: 4) EPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGR

The short form of the plasmin heavy chain A is 483 amino acids. Theamino acid sequence of the short form of the plasmin heavy chain A is asfollows:

(SEQ ID NO: 5) VYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGR

The amino acid sequence of the activation peptide comprises thefollowing amino acid sequence:

(SEQ ID NO: 6) EPLDDYVNIQGASLFSVIKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKK

The plasmin light chain B is 230 amino acids. The amino acid sequence ofthe plasmin light chain B is as follows:

(SEQ ID NO: 7) VVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN

In some preferred embodiments, the plasmin can be a miniplasmin or amicroplasmin or a derivative thereof. Miniplasmin and microplasmin areproduced upon the activation of miniplasminogen and microplasminogen byplasminogen activators such as, but not limited to, streptokinase,staphylokinase, tissue-type plasminogen activator or urokinase.Miniplasminogen and microplasminogen are derived from plasminogen, whichis a single chain glycoprotein that is an important component ofmammalian blood. Human plasminogen is a multi-domain protein of 791residues (SEQ ID NO:1), composed of an N-terminal pre-activation domain,five homologous kringle domains each of about 80 amino acids, a serineprotease catalytic domain and inter-domain connecting sequences. Plasminor plasminogen activators cleave the peptide bonds between Arg-68 andMet-69, or Lys-77 and Lys-78 or Lys-78 and Val-79 at the N-terminal ofhuman plasminogen, resulting in shorter proenzymes calledLys-plasminogens (for example, proteins consisting of amino acids 69-791or 78-791 or 79-791). Additional cleavage by the enzyme elastase removesthe first four kringle domains producing the proenzyme, miniplasminogen(typically amino acids 442-791). Further cleavage of the fifth kringleyields the proenzyme, microplasminogen (typically amino acids 543-791).The kringles of plasminogen contain lysine-binding sites that mediatespecific binding of plasminogen to substrates such as fibrin. Theproenzyme forms of plasminogen are activated to their enzymaticallyactive form by the cleavage of the peptide bond between Arg-561 andVal-562 to yield a disulfide bonded double chain form of thecorresponding protein. The product of activation of a plasminogenprotein is called a plasmin Thus, the product of Lys-plasminogenactivation is called Lys-plasmin, while the products of activation ofminiplasminogen and microplasminogen, are referred to as miniplasmin andmicroplasmin, respectively. Like plasmin, miniplasmin and microplasminpossess catalytic activity. An advantage of miniplasmin and microplasminover plasmin is their smaller size compared to plasmin Thus, bothmicroplasmin and miniplasmin are expected to have faster diffusion ratesin the vitreous than plasmin.

The plasmin of the present invention may comprise any one of theplasminogen-related sequences described herein, for example, any one ofSEQ ID NOs: 1 and 3-7, or a functional fragment or variant thereof.

The plasmin may also be ocriplasmin (JETREA®) or variants or derivativesthereof. Ocriplasmin is a recombinant truncated form of human plasminproduced by recombinant DNA technology in a Pichia pastoris expressionsystem. Ocriplasm is a protein made up of 249 amino acids with amolecular weight of 27.2 kDa, and has two peptide chains. The amino acidsequence for the truncated heavy chain is as follows:

(SEQ ID NO: 8) APSFDCGKPQVEPKKCPGRThe amino acid sequence for the light chain is as follows:

(SEQ ID NO: 9) VVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNNThe present invention further encompasses variants and derivatives ofocriplasmin.

The plasmin may be isolated from blood using known isolation techniques.Alternatively, plasminogen may be isolated from the blood using knownisolation techniques, and then incubated with proteases that cleaveplasminogen into active plasmin to produce purified or isolated plasminsuitable for use in the methods described herein.

The plasmin may be synthesized chemically using commercially availablepeptide synthesizers to produce purified or isolated plasmin suitablefor use in the methods described herein. Chemical synthesis of peptidesand proteins can be used for the incorporation of modified or unnaturalamino acids, including D-amino acids and other small organic molecules.Replacement of one or more L-amino acids in a peptide or protein withthe corresponding D-amino acid isoforms can be used to increaseresistance to enzymatic hydrolysis, and to enhance one or moreproperties of biological activity, i.e., functional potency or durationof action. Other modifications to the plasmin can be introduced, forexample, cross-linking to change the conformation to alter the potency,selectivity or stability of the plasmin.

For example, the plasmin may be purchased from a commercial vendor, suchas Sigma Aldrich, Catalog Number P1867. The present invention alsoencompasses variants or derivatives of the plasmin supplied by SigmaAldrich (Cat. No. P1867).

The plasmin may be a recombinant plasmin obtained by methods well knownin the art for recombinant protein expression and purification. A DNAmolecule encoding a plasmin or a variant or analog thereof can begenerated from known DNA sequences or by deducing the nucleic acidsequences from the amino acid sequence based on known codon usage. TheDNA molecule encoding a plasmin can be cloned into a suitable vector,such as a cloning or expression vector, by any of the methodologiesknown in the art. The cloning or expression vectors contain all thecomponents required for additional cloning of the plasmin DNA, such asrestriction enzyme sites, or for the expression of the plasmin, such asa host-specific promoter, and optionally, enhancer sequences. Theexpression vector can be introduced and expressed in a host cell. A hostcell can be any prokaryotic or eukaryotic cell. For example, the plasmincan be expressed in bacterial cells (i.e., E. coli), yeast, insect cells(i.e., Sf9), or mammalian cells. Other suitable host cells are known tothose skilled in the art. The host cell can be used to produce oroverexpress the plasmin, variant or derivative thereof in culture. Thenthe biologically expressed plasmin, variant or derivative thereof may bepurified using known purification techniques, such as affinitychromatography, to produce purified or isolated plasmin suitable for usein the methods described herein.

In some embodiments, a variant, derivative or analog of a plasmin may bepreferred. Variants, derivatives and analogs of plasmin can beidentified or generated by one ordinarily skilled in the art. Theplasmin variants, derivatives and analogs can be generated, for example,by using the recombinant methods or methods of synthesis describedherein. Plasmin derivatives known in the art include miniplasmin andmicroplasmin are also suitable for use in the methods disclosed herein.

As used herein, the term “derivative” and “variant” may be usedinterchangeably and refers to a plasmin that differs from naturallyoccurring plasmin, but retains the essential properties thereof. Forexample, the plasmin derivative may be a biologically active fragment ofplasmin, for example, a truncated plasmin. The biologically activefragment contains the catalytic domain of plasmin and possesses serineprotease catalytic activity. Alternatively, the plasmin derivative maybe a mutated plasmin, wherein at least 1 amino acid, at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 20, at least 30, at least 40, or at least50 amino acids are mutated from the wild-type plasmin. Mutated plasminmay have altered sequences by substitutions, additions, or deletions,that still result in functionally equivalent molecules. In oneembodiment, the plasmin derivative is about 99%, about 98%, about 97%,about 96%, about 95%, about 90%, about 85%, about 80%, about 75%, about70%, about 65%, about 60%, about 55%, or about 50% identity to wild-typeplasmin. In some instances, the mutation may increase the potency(protease activity), stability, or number of targets of the plasmin. Inanother aspect, the mutation may decrease the potency, stability, ornumber of targets of the plasmin. The mutation may be a conservativeamino acid substitution. Alternatively, the mutation may be anon-conservative amino acid substitution. In another embodiment, theplasmin derivative may be chemically modified, for example, by theaddition of a chemical moiety that alters activity or stability.

The term “% identity,” in the context of two or more nucleic acid orpolypeptide sequences, refer to two or more sequences or subsequencesthat are the same or have a specified percentage of amino acid residuesor nucleotides that are the same, when compared and aligned for maximumcorrespondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. For example, % identityis relative to the entire length of the coding regions of the sequencesbeing compared.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters. Percent identity is determined usingsearch algorithms such as BLAST and PSI-BLAST (Altschul et al., 1990, JMol Biol 215:3, 403-410; Altschul et al., 1997, Nucleic Acids Res 25:17,3389-402).

The term “analog” as used herein refers to compounds or peptides thatretain the essential protease activity of the plasmin. For example, theanalog may bear some amino acid sequence similarity to or no sequencesimilarity to naturally occurring plasmin. However all plasmin analogsretain the functional capability of protease cleavage of any one of thetargets of plasmin (i.e., laminin, fibrin), and/or the preferentialcleavage at the carboxyl side of Lysine and Arginine residues.

Plasmin derivatives and analogs can be identified by one ordinarilyskilled in the art by screening combinatorial libraries of mutants ofplasmin or general therapeutic or pharmaceutical compounds.

Methods of Use

The present invention provides methods for enhancing the delivery of atherapeutic agent to the eye. The methods of the present inventioninclude administering a plasmin and a therapeutic agent to the eye. Thetherapeutic agent may be delivered to the eye by any method known in theart. Routes of administration include, but are not limited to,intravitreal, intracameral, subconjunctival, subtenon, retrobulbar,posterior juxtascleral, or topical. Delivery methods include, forexample, injection by a syringe and a drug delivery device, such as animplanted vitreal delivery device (i.e., VITRASERT®).

Preferably, the therapeutic agent is administered to the vitreous byintravitreal injection for delivery of therapeutic agents to the retina.In some embodiments, the methods of the present invention provideenhanced delivery to cells of the retina. Exemplary retinal cells,include, but are not limited to, photoreceptor cells (e.g., rods, cones,and photosensitive retinal ganglion cells), horizontal cells, bipolarcells, amacrine cells, retinal ganglion cells, Müller glial cell, andretinal pigment epithelial cells.

In one embodiment, the plasmin or derivative thereof is administeredconcurrently or sequentially with the therapeutic agent. For concurrentadministration, the plasmin or derivative thereof can be formulated withthe therapeutic agent in a single composition suitable for delivery, forexample, injection, by methods known in the art. Alternatively, theplasmin or derivative thereof can be injected in separate compositions,simultaneously or sequentially. In a preferred embodiment, the plasminmay be administered prior to administration of the therapeutic agent.

Such formulations comprise a pharmaceutically and/or physiologicallyacceptable vehicle, diluent, carrier or excipient, such as bufferedsaline or other buffers, e.g., HEPES, to maintain physiologic pH. For adiscussion of such components and their formulation, see, generally,Gennaro, A E., Remington: The Science and Practice of Pharmacy,Lippincott Williams & Wilkins Publishers; 2003 or latest edition). Seealso, WO00/15822. If the preparation is to be stored for long periods,it may be frozen, for example, in the presence of glycerol.

The dosage of plasmin or derivative thereof to be administered can beoptimized by one of ordinary skill in the art. Delivery to certaintarget ocular tissues may require lower doses of plasmin or higher dosesof plasmin, depending on the location of the target tissue, interveningocular structures, and ability of the agent to penetrate the targettissue. Preferably, the dose of plasmin administered is about 0.001 UI(enzyme units) per eye, 0.025 UI per eye, about 0.05 UI per eye, about0.075 UI per eye, about 0.100 UI per eye, about 0.150 UI per eye, orabout 0.200 UI per eye.

In some embodiments, the methods for enhanced delivery disclosed hereinmay provide increased efficacy of a therapeutic agent. Increasedefficacy of the therapeutic agent can be determined by measuring thetherapeutic effect of the therapeutic agent. Treatment is efficacious ifthe treatment leads to clinical benefit such as, alleviation of asymptom in the subject. For example, in a degenerative retinal disease,such as retinitis pigmentosa, treatment is efficacious when lightsensitivity or another aspect of vision is improved or restored. Whentreatment is applied prophylactically, “efficacious” means that thetreatment retards or prevents an ocular disease or disorder or preventsor alleviates a symptom of clinical symptom of an ocular disease ordisorder. Efficaciousness is determined in association with any knownmethod for diagnosing or treating the particular ocular disease ordisorder.

In some embodiments, the therapeutic agent is a nucleic acid or anucleic acid expression vector (i.e. a viral vector) encoding atherapeutic transgene or an siRNA species (i.e., short hairpin ormicroRNA). With regard to such therapeutic agents, the enhanced deliveryof a therapeutic agent provided by the methods disclosed herein mayresult in increased transduction efficiency. Increased transductionefficiency can be determined by measuring the level of expression of thetransgene introduced by the viral vector.

Therapeutic Agents

The therapeutic agents to be delivered to the eye by the methodsdescribed herein are any therapeutic agents known in the art fortreating, alleviating, reducing, or preventing a symptom of an oculardisease, an ocular disorder, or an ocular condition. The therapeuticagent may be a small molecule, a nucleic acid, an antibody, or apeptide.

Examples of small molecules suitable for use in the methods describedherein include, but are not limited to, tyrosine kinase inhibitors,antibiotics, anti-inflammatory agents, glucocorticoids, opioidantagonists, and other enzyme inhibitors.

Examples of nucleic acids suitable for use in the methods describedherein include, but are not limited to, viral vectors encodingtherapeutic transgenes (i.e., channelopsins, or halorhodopsin), RNAinterference molecules (i.e., short hairpins, siRNA, or microRNAs). In aparticularly preferred embodiment, the therapeutic agents are viralvectors encoding transgenes for gene therapy. Particularly preferredviral vectors are rAAV vectors that encode channelopsins orhalorhodopsins for expression in the retina to restore lightsensitivity.

Examples of antibodies suitable for use in the methods described hereininclude, but are not limited to, ranibizumab (Lucentis®), VEGFantibodies (Eylea®), bevacizumab (Avastin®), infliximab, etanercept, andadalimumab.

Examples of proteins or peptides suitable for use in the methodsdescribed herein include, but are no limited to, microplasmin(Ocriplasmin, Jetrea®), macugen pagylated polypeptide (Pegaptanib), andintegrin peptides. In some aspects, the peptide therapeutic is acollection of peptides, containing two or more peptides.

Any of the therapeutic agents described herein may be optionallyencapsulated in a carrier, such as a nanoparticle, a polymer, or aliposome. These carrier agents may serve to further enhance the deliveryof the therapeutic agent to the eye. In some aspects, the carrier agentsmay alter the properties of the therapeutic agents, such as increasingthe stability (half-life) or providing sustained-release properties tothe therapeutic agents. Alternatively, the carrier may protect thetherapeutic agent from the proteolytic activities of plasmin ifformulated in the same composition for delivery.

Gene Therapy

As a large number of ocular diseases and disorders result from aberrantgene expression in various ocular tissues, gene therapy possessesincreasing potential as an effective therapy. However, the efficacy ofgene therapy in the eye has been limited due to the challenges ofeffective delivery and transduction of the therapeutic viral vectorsthroughout any ocular tissue.

Thus, the present invention provides methods for increased efficiency ofdelivery of transgenes to the eye for treating an ocular disease ordisorder, or for restoring or improving vision. Transgenes of particularinterest for restoration of photosensitivity or vision includephotosensitive proteins, such as opsin genes or rhodopsin genes. As usedherein, “transgene” refers to a polynucleotide encoding a polypeptide ofinterest, wherein the polynucleotide is present in a nucleic acidexpression vector suitable for gene therapy (e.g., a viral vector suchas AAV).

Previous studies have shown that injection of a recombinantadeno-associated viral vector encoding a transgene, such aschannelopsin-2, results in poor delivery of the vector and lowexpression of Chop2 in the inner retinal cells, especially bipolarcells. In non-human primates, AAV-mediated gene transfection was foundto be more efficient in peripheral retina, fovea, and along bloodvessels, suggesting that inner limiting membrane (ILM), which is theboundary between the retina and the vitreous space, is a major barrier(Ivanova et al., 2010).

The present invention provides a solution to this problem by usingplasmin or derivatives thereof to dissolve the components the ILM, suchas laminin and fibronectin. Accordingly, therapeutic agents will havegreater accessibility to the retina, specifically the cells of the innerretina such as the retinal bipolar cells, retinal ganglion cells, Müllerglial cells, and retinal pigment epithelial cells. The methods describedherein provide enhanced delivery of therapeutic compounds, such astherapeutic viral vectors. The enhanced delivery of viral vectors isdemonstrated by increased transduction efficiency, increased expressionof the therapeutic transgene (i.e., Chop2), and increased efficacy ofthe therapeutic compound (i.e., increased light sensitivity orrestoration of vision).

Nucleic acid expression vectors suitable for use in gene therapy areknown in the art. For example, the nucleic acid expression vector is aviral vector. The viral vectors can be retroviral vectors, adenoviralvectors, adeno-associated vectors (AAV), or lentiviral vectors, or anyengineered or recombinant viral vector known in the art. Particularlypreferred viral vectors are adeno-associated vectors, for example,AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10,AAV-11, AAV-12 or any engineered or recombinant AAV known in the art. Ina particularly preferred embodiment, the vector is recombinant AAV-2(rAAV2).

In some embodiments, a recombinant adeno-associated viral (rAAV) vectorcomprises a capsid protein with a mutated tyrosine residue which enablesto the vector to have improved transduction efficiency of a target cell,e.g., a retinal bipolar cell (e.g. ON or OFF retinal bipolar cells; rodand cone bipolar cells). In some cases, the rAAV further comprises apromoter (e.g., mGluR6, or fragment thereof) capable of driving theexpression of a protein of interest in the target cell.

In one embodiment, a mutation may be made in any one or more oftyrosine: residues of the capsid protein of AAV 1-12 or hybrid. AAVs. Inspecific embodiments these are surface exposed tyrosine residues. In arelated embodiment the tyrosine residues are part of the VP1, VP2, orVP3 capsid protein. In exemplary embodiments, the mutation may be madeat one or more of the following amino acid residues of an AAV-VP3 capsidprotein: Tyr252, Tyr272, Tyr444, Tyr500, Tyr700, Tyr704, Tyr730; Tyr275,Tyr281, Tyr508, Tyr576, Tyr612, Tyr673 or Tyr720. Exemplary mutationsare tyrosine-to-phenylalanine mutations including, but not limited to,Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F, Y281F, Y508F,Y576F, Y612G, Y673F and Y720F. in a specific embodiment these mutationsare made in the AAV2 serotype. In some cases, an AAV2 serotype comprisesa Y444F mutation and/or an AAV8 serotype comprises a Y733F mutation,wherein 444 and 733 indicate the location of a point tyrosine mutationof the viral capsid. In further embodiments, such mutated AAV2 and AAV8serotypes encode a light-sensitive protein and also comprise a modifiedmGluR6 promoter to drive expression of such light-sensitive protein.Such AAV vectors are described in, for example, Petrs-Silva et al., MolTher., 2011 19:293-301).

In some embodiments, the expression of the therapeutic transgene isdriven by a constitutive promoter, i.e., CAG promoter, CMV promoter,LTR. In other embodiments, the promoter is an inducible or acell-specific promoter. Cell type-specific promoters that enabletransgene expression in specific subpopulations of cells, i.e., retinalneuron cells or degenerating cells, may be preferred. These cells mayinclude, but are not limited to, a retinal ganglion cell, aphotoreceptor cell, a bipolar cell, a rod bipolar cell, an ON-type conebipolar cell, a retinal ganglion cell, a photosensitive retinal ganglioncell, a horizontal cell, an amacrine cell, an AII amacrine cell, or aretinal pigment epithelial cell. Cell type-specific promoters are wellknown in the art. Particularly preferred cell type-specific promotersinclude, but are not limited to mGluR6, NK-3, and Pcp2(L7). Celltype-specific promoters modified using recombinant DNA techniques knownin the art to increase efficiency of expression and selective targetingare also encompassed in the present invention. For example, a modifiedmGluR6 promoter contains a combination of regulatory elements from themGluR6 gene, as described in U.S. Provisional Application No.61/951,360, hereby incorporated by reference in its entirety.

In one embodiment of the present invention, the therapeutic transgenecan be any light-sensitive opsin. The opsin family of genes includesvertebrate (animal) and invertebrate opsins. Animal opsins are G-proteincoupled receptors (GPCRs) with 7-transmembrane helices which regulatethe activity of ion channels. Invetertebrate rhodopsins are usually notGPCRs but are light-sensitive or light-activated ion pumps or ionchannels.

As referred to herein, an opsin gene or light-sensitive proteinincludes, but is not limited to, channelrhodopsins, or channelopsins,(i.e., ChR1, ChR2, vChR1 from Volvox carteri, vChR2, and other variantsidentified from any vertebrate, invertebrate, or microbe),halorhodopsins (NpHR), melanopsins, pineal opsins, photopsins,bacteriorhodopisins, proteorhodopsins and functional variants orchimeras thereof. A light-sensitive protein of this invention can occurnaturally in plant, animal, archaebacterial, algal, or bacterial cells,or can alternatively be created through laboratory techniques. Examplesof opsin genes are discussed in further detail below.

Examples of channelrhodopsins, or channelopsins, as transgenes in thepresent invention include channelrhodopsins Chop1 (also known as ChR1)(GenBank accession number AB058890/AF385748) and Chop2 (also known asChR2) (GenBank accession number AB058891/AF461397) are two rhodopsinsfrom the green alga Chlamydomonas reinhardtii (Nagel, 2002; Nagel, 2003)Channelopsins are a seven transmembrane domain proteins that becomephoto-switchable (light sensitive) when bound to the chromophoreall-trans-retinal. Channelopsins, when linked to a retinal molecule viaSchiff base linkage forms a light-gated, nonspecific, inwardlyrectifying, cation channel, called a channelrhodopsin. Theselight-sensitive channels that, when expressed and activated in neuraltissue, allow for a cell to be depolarized when stimulated with light(Boyden, 2005). A Chop2 fragment (315 amino acids) has been shown toefficiently increase photosensitivity and vision in mouse models ofphotoreceptor degeneration (Bi et al., Neuron, 2006, and U.S. Pat. No.8,470,790; both of which are hereby incorporated by reference). Chop2mutants and variants as described in PCT Publication WO 2013/134295(hereby incorporated by reference) may also be expressed using thepromoters described herein. The present invention also provides for useof Volvox carteri channelrhodopsins (i.e., vChR1 and vChR2).

NpHR (Halorhodopsin) (GenBank accession number EF474018) is from thehaloalkaliphilic archaeon Natronomonas pharaonic. In certain embodimentsvariants of NpHR can be created. In specific embodiments single ormultiple point mutations to the NpHR protein can result in NpHRvariants. In specific embodiments a mammalian codon optimized version ofNpHR can be utilized. In one embodiment NpHR variants are utilized. Inone specific embodiment eNpHR (enhanced NpHR) is utilized. Addition ofthe amino acids FCYENEV to the NpHR C-terminus along with the signalpeptide from the (3 subunit of the nicotinic acetylcholine receptor tothe NpHR N-terminus results in the construction of eNpHR.

Melanopsin (GenBank accession number 6693702) is a photopigment found inspecialized photosensitive ganglion cells of the retina that areinvolved in the regulation of circadian rhythms, pupillary light reflex,and other non-visual responses to light. In structure, melanopsin is anopsin, a retinylidene protein variety of G-protein-coupled receptor.Melanopsin resembles invertebrate opsins in many respects, including itsamino acid sequence and downstream signaling cascade. Like invertebrateopsins, melanopsin appears to be a bistable photopigment, with intrinsicphotoisomerase activity. In certain embodiments variants of melanopsincan be created. In specific embodiments single or multiple pointmutations to the melanopsin protein can result in melanopsin variants.

Light-sensitive proteins may also include proteins that are at leastabout 10%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, or at leastabout 99% identical to any of the light-sensitive proteins describedherein (i.e., ChR1, ChR2, vChR1, vChR2, NpHR and melanopsin). Thelight-sensitive proteins of the present invention may also includeproteins that have at least one mutation. The mutation may be a pointmutation.

In some embodiments, light-sensitive proteins can modulate signalingwithin neural circuits and bidirectionally control behavior of ionicconductance at the level of a single neuron. In some embodiments theneuron is a retinal neuron, a retinal bipolar cell (e.g. ON or OFFretinal bipolar cells; rod and cone bipolar cells), a retinal ganglioncell, a photoreceptor cell, or a retinal amacrine cell.

In some embodiments, a polyA tail can be inserted downstream of thetransgene in an expression cassette or nucleic acid expression vector ofthe present invention. Suitable polyA tails are known in the art, andinclude, for example, human growth hormone poly A tail (hGHpA), bovinegrowth hormone polyA tail (bGHpA), bovine polyA, SV40 polyA, and AV40pA.

Upon illumination by the preferred dose of light radiation, rhodopsinproteins opens the pore of the channel, through which H⁺, Na⁺, K⁺,and/or Ca²⁺ ions flow into the cell from the extracellular space.Activation of the rhodopsin channel typically causes a depolarization ofthe cell expressing the channel Depolarized cells produce gradedpotentials and or action potentials to carry information from therhodopsin-expressing cell to other cells of the retina or brain, toincrease light sensitivity or restore vision. Methods of improvingvision or light sensitivity by administration of a vector encoding achannelopsin (or variant thereof) are described in PCT/US2007/068263,the contents of which are herein incorporated in its entirety.

Accordingly, a dual rhodopsin system can be used to recapitulate the ONand OFF pathways integral to visual processing and acuity. Briefly, aChop2 protein of the present invention can be specifically targeted toON type retinal neurons (i.e., ON type ganglion cells and/or ON typebipolar cells), while a hypopolarizing light sensor (i.e., halorhodopsinor other chloride pump known in the art) can be targeted to OFF typeretinal neurons (i.e. OFF type ganglion cells and/or OFF type bipolarcells) to create ON and OFF pathways. The specific targeting topreferred cell subpopulations can be achieved through the use ofdifferent cell type-specific promoters. For example, Chop2 expressionmay be driven by the mGluR6 promoter for targeted expression in ON-typeretinal neurons (i.e., ON type ganglion cells and/or ON type bipolarcells) while a hypopolarizing channel, such as halorhodopsin, expressionis driven by the NK-3 promoter for targeted expression in OFF-typeretinal neurons (i.e., OFF type ganglion cells and/or OFF type bipolarcells).

An alternative approach to restore ON and OFF pathways in the retina isachieved by, expressing a depolarizing light sensor, such as ChR2, torod bipolar cells or AII amacrine. In this approach, the depolarizationof rod bipolar cells or AII amacrine cells can lead to the ON and OFFresponses at the levels of cone bipolar cells and the downstream retinalganglion cells. Thus, the ON and OFF pathways that are inherent in theretina are maintained.

An effective amount of rAAV virions carrying a nucleic acid sequenceencoding the rhodopsin DNA under the control of the promoter of choice,preferably a constitutive CMV promoter or a cell-specific promoter suchas mGluR6, is preferably in the range of between about 10¹⁰ to about10¹³ rAAV infectious units in a volume of between about 25 and about 800μl per injection. The rAAV infectious units can be measured according toMcLaughlin, S K et al., 1988, J Virol 62:1963. More preferably, theeffective amount is between about 10¹⁰ and about 10¹² rAAV infectiousunits and the injection volume is preferably between about 50 and about150 μl. Other dosages and volumes, preferably within these ranges butpossibly outside them, may be selected by the treating professional,taking into account the physical state of the subject (preferably ahuman), who is being treated, including, age, weight, general health,and the nature and severity of the particular ocular disorder.

It may also be desirable to administer additional doses (“boosters”) ofthe present nucleic acid(s) or rAAV compositions. For example, dependingupon the duration of the transgene expression within the ocular targetcell, a second treatment may be administered after 6 months or yearly,and may be similarly repeated. Neutralizing antibodies to AAV are notexpected to be generated in view of the routes and doses used, therebypermitting repeat treatment rounds.

The need for such additional doses can be monitored by the treatingprofessional using, for example, well-known electrophysiological andother retinal and visual function tests and visual behavior tests. Thetreating professional will be able to select the appropriate testsapplying routine skill in the art. It may be desirable to inject largervolumes of the composition in either single or multiple doses to furtherimprove the relevant outcome parameters.

Ocular Disorders

The ocular disorders for which the methods of the present invention areintended and may be used to improve one or more parameters of visioninclude, but are not limited to, developmental abnormalities that affectboth anterior and posterior segments of the eye. Anterior segmentdisorders include glaucoma, cataracts, corneal dystrophy, keratoconus.Posterior segment disorders include blinding disorders caused byphotoreceptor malfunction and/or death caused by retinal dystrophies anddegenerations. Retinal disorders include congenital stationary nightblindness, age-related macular degeneration, congenital conedystrophies, and a large group of retinitis-pigmentosa (RP)-relateddisorders. These disorders include genetically pre-disposed death ofphotoreceptor cells, rods and cones in the retina, occurring at variousages. Among those are severe retinopathies, such as subtypes of RPitself that progresses with age and causes blindness in childhood andearly adulthood and RP-associated diseases, such as genetic subtypes ofLCA, which frequently results in loss of vision during childhood, asearly as the first year of life. The latter disorders are generallycharacterized by severe reduction, and often complete loss ofphotoreceptor cells, rods and cones. Other ocular diseases that maybenefit from the methods described herein include, but are not limitedto, retinoblastoma, ocular melanoma, diabetic retinopathy, hypertensiveretinopathy, any inflammation of the ocular tissues (i.e., chorioretinalinflammation, scleritis, keratitis, uveitis, etc.), or infection (i.e.,bacterial or viral).

In particular, the viral-mediated delivery of rhodopsins using themethods of the present invention useful for the treatment and/orrestoration of at least partial vision to subjects that have lost visiondue to ocular disorders, such as RPE-associated retinopathies, which arecharacterized by a long-term preservation of ocular tissue structuredespite loss of function and by the association between function lossand the defect or absence of a normal gene in the ocular cells of thesubject. A variety of such ocular disorders are known, such as childhoodonset blinding diseases, retinitis pigmentosa, macular degeneration, anddiabetic retinopathy, as well as ocular blinding diseases known in theart. It is anticipated that these other disorders, as well as blindingdisorders of presently unknown causation which later are characterizedby the same description as above, may also be successfully treated bythe methods described herein. Thus, the particular ocular disordertreated by the present invention may include the above-mentioneddisorders and a number of diseases which have yet to be socharacterized.

Restoration of Light Sensitivity

These methods described herein may be used in subjects of normal and/orimpaired vision. The enhanced delivery of a therapeutic compound, asdescribed herein, may preserve, improve, or restore vision. The term“vision” as used herein is defined as the ability of an organism tousefully detect light as a stimulus for differentiation or action.Vision is intended to encompass the following:

-   -   1. Light detection or perception—the ability to discern whether        or not light is present;    -   2. Light projection—the ability to discern the direction from        which a light stimulus is coming;    -   3. Resolution—the ability to detect differing brightness levels        (i.e., contrast) in a grating or letter target; and    -   4. Recognition—the ability to recognize the shape of a visual        target by reference to the differing contrast levels within the        target.        Thus, “vision” includes the ability to simply detect the        presence of light. The methods of the present invention can be        used to improve or restore vision, wherein the improvement or        restoration in vision includes, for example, increases in light        detection or perception, increase in light sensitivity or        photosensitivity in response to a light stimulus, increase in        the ability to discern the direction from which a light stimulus        is coming, increase in the ability to detect differing        brightness levels, increase in the ability to recognize the        shape of a visual target, and increases in visual evoked        potential or transmission from the retina to the cortex. As        such, improvement or restoration of vision may or may not        include full restoration of sight, i.e., wherein the vision of        the patient treated with the present invention is restored to        the degree to the vision of a non-affected individual. The        visual recovery described in the animal studies described below        may, in human terms, place the person on the low end of vision        function by increasing one aspect of vision (i.e., light        sensitivity, or visual evoked potential) without restoring full        sight. Nevertheless, placement at such a level would be a        significant benefit because these individuals could be trained        in mobility and potentially in low order resolution tasks which        would provide them with a greatly improved level of visual        independence compared to total blindness. Even basic light        perception can be used by visually impaired individuals, whose        vision is improved using the present compositions and methods,        to accomplish specific daily tasks and improve general mobility,        capability, and quality of life.

The degree of restoration of vision can be determined through themeasurement of vision before, and preferably after, administering avector comprising, for example, DNA encoding a therapeutic transfenesuch as Chop2 or halorhodopsin or both. Vision can be measured using anyof a number of methods well-known in the art or methods not yetestablished. Vision, as improved or restored by the present invention,can be measured by any of the following visual responses:

-   -   1. a light detection response by the subject after exposure to a        light stimulus—in which evidence is sought for a reliable        response of an indication or movement in the general direction        of the light by the subject individual when the light it is        turned on;    -   2. a light projection response by the subject after exposure to        a light stimulus in which evidence is sought for a reliable        response of indication or movement in the specific direction of        the light by the individual when the light is turned on;    -   3. light resolution by the subject of a light vs. dark patterned        visual stimulus, which measures the subject's capability of        resolving light vs dark patterned visual stimuli as evidenced        by:        -   a. the presence of demonstrable reliable optokinetically            produced nystagmoid eye movements and/or related head or            body movements that demonstrate tracking of the target (see            above) and/or        -   b. the presence of a reliable ability to discriminate a            pattern visual stimulus and to indicate such discrimination            by verbal or non-verbal means, including, for example            pointing, or pressing a bar or a button; or    -   4. electrical recording of a visual cortex response to a light        flash stimulus or a pattern visual stimulus, which is an        endpoint of electrical transmission from a restored retina to        the visual cortex, also referred to as the visual evoked        potential (VEP). Measurement may be by electrical recording on        the scalp surface at the region of the visual cortex, on the        cortical surface, and/or recording within cells of the visual        cortex.

Thus, improvement or restoration of vision, according to the presentinvention, can include, but is not limited to: increases in amplitude orkinetics of photocurents or electrical response in response to lightstimulus in the retinal cells, increases in light sensitivity (i.e.,lowering the threshold light intensity required for initiating aphotocurrent or electrical response in response to light stimulus,thereby requiring less or lower light to evoke a photocurrent) of theretinal cells, increases in number or amplitude of light-evoked spikingor spike firings, increases in light responses to the visual cortex,which includes increasing in visual evoked potential transmitted fromthe retina or retinal cells to the visual cortex or the brain.

Both in vitro and in vivo studies to assess the various parameters ofthe present invention may be used, including recognized animal models ofblinding human ocular disorders. Large animal models of humanretinopathy, e.g., childhood blindness, are useful. The examplesprovided herein allow one of skill in the art to readily anticipate thatthis method may be similarly used in treating a range of retinaldiseases.

While earlier studies by others have demonstrated that retinaldegeneration can be retarded by gene therapy techniques, the presentinvention demonstrates a definite physiological recovery of function,which is expected to generate or improve various parameters of vision,including behavioral parameters.

Behavioral measures can be obtained using known animal models and tests,for example performance in a water maze, wherein a subject in whomvision has been preserved or restored to varying extents will swimtoward light (Hayes, J M et al., 1993, Behav Genet 23:395-403).

In models in which blindness is induced during adult life or congenitalblindness develops slowly enough that the individual experiences visionbefore losing it, training of the subject in various tests may be done.In this way, when these tests are re-administered after visual loss totest the efficacy of the present compositions and methods for theirvision-restorative effects, animals do not have to learn the tasks denovo while in a blind state. Other behavioral tests do not requirelearning and rely on the instinctiveness of certain behaviors. Anexample is the optokinetic nystagmus test (Balkema G W et al., 1984,Invest Ophthalmol Vis Sci. 25:795-800; Mitchiner J C et al., 1976,Vision Res. 16:1169-71).

The present invention may also be used in combination with other formsof vision therapy known in the art to improve or restore vision. Forexample, the use of visual prostheses, which include retinal implants,cortical implants, lateral geniculate nucleus implants, or optic nerveimplants. Thus, in addition to genetic modification of surviving retinalneurons using the present methods, the subject being treated may beprovided with a visual prosthesis before, at the same time as, or afterthe molecular method is employed. The effectiveness of visualprosthetics can be improved with training of the individual, thusenhancing the potential impact of the Chop2 transformation of patientcells as contemplated herein. Training methods, such as habituationtraining characterized by training the subject to recognize recognize(i) varying levels of light and/or pattern stimulation, and/or (ii)environmental stimulation from a common light source or object as wouldbe understood by one skilled in the art; and orientation and mobilitytraining characterized by training the subject to detect visually localobjects and move among said objects more effectively than without thetraining. In fact, any visual stimulation techniques that are typicallyused in the field of low vision rehabilitation are applicable here.

As used herein, by a “subject” is meant an individual. Thus, the“subject” can include domesticated animals (e.g., cats, dogs, etc.),livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratoryanimals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds.“Subject” can also include a mammal, such as a primate or a human.Preferably, the subject is a human. A “subject in need thereof” is asubject suffering from or at risk of developing or suffering from anocular disease or disorder. A subject at risk of developing or sufferingfrom an ocular disease or disorder can be diagnosed by a physician orocular specialist using routine methods in the art.

EXAMPLES Example 1: Plasmin Increases Delivery of AAV2-Vector EncodingChop2 to the Retina

Delivery of a therapeutic viral construct encoding a functionalGFP-channelopsin-2 protein to the retina was examined Injection of6×10¹² vg/ml of AAV2 vector AAV2/2-ChR2-GFP-WPRE-hGHpA in control (FIG.1A) or co-injection with plasmin (FIG. 1B) was performed into thevitreous space of the eye of one month old C56BL/6J. The concentrationof plasmin injected with vector was 0.025 IU/eye. After one month, themice retinas were isolated, and retinal vertical sections were prepared.The sections were immunostained and cells were countedImmunofluorescence analysis of the sections showed that co-injectionwith plasmin increased the transduction efficiency of the therapeuticAAV2-ChR2-GFP vector, as evidenced by increased fluorescence incomparison to control (FIG. 1).

Example 2: Plasmin Increases Transduction Efficiency in Retinal GanglionCells

Using the same experimental set-up as in Example 1, a vector (2×10¹²vg/ml) encoding GFP was co-injected with either control or varyingconcentrations of plasmin: low (L=0.005 IU/eye), middle (M=0.025IU/eye), and high (H=0.100 IU/eye). After 1 month, retinal whole mountswere prepared and immunostained. Representative images for each plasminconcentration and control are shown in FIG. 2. As shown, treatment withplasmin increases GFP expression.

To further quantify these results, GFP-expressing retinal ganglion cellswere counted from multiple unit areas of 223 μm×167 μm. The results arepresented in FIG. 3A. As shown, treatment with low, middle and highdoses of plasmin resulted in statistically significantly increasedlevels of GFP expression in retinal ganglion cells.

Neurotoxicity as a result of plasmin injection was also examined. Theretinal whole mounts were stained with DAPI for cell-counting. Thenumber of cells over multiple unit areas of 223 μm×167 μm were countedand compared between control and low, middle and high doses of plasmin.As shown in FIG. 3B, the cell counts were not found to differsignificantly between control and plasmin-treated retinas (p=0.74). Assuch, the tested concentrations of plasmin were not shown to have anyneurotoxic effect to the retinal ganglion cells, thereby indicating thatplasmin is safe for use in the eye, even at high doses.

Example 3: Plasmin Increases Transduction Efficiency in Retinal BipolarCells

Comparison of the transduction efficiency of a viral vector encodingmCherry fluorescent protein when co-injected with differentconcentrations of plasmin was assessed in vivo. Specifically, overalllevels and the localization of mCherry expression throughout the retinawere examined. An AAV2 vector with an Y444F capsid mutation carryingmCherry under control of an mGluR6 promoter were injected at aconcentration of 2×10¹² vg/ml. The mGluR6 promoter directs expression ofmCherry specifically to the retinal bipolar cells.

The AAV2 mCherry vector was co-injected with three doses of plasmin,high (H=0.100 IU/eye), middle (M=0.025 IU/eye), and low (L=0.005IU/eye). After 1 month, the retinas were isolated and retinalwhole-mounts were prepared. Transduction efficiency was evaluated byimmunostaining of mCherry for immunofluorescence analysis and cellcounting. Cells were counted from multiple unit areas of 223 μm×167 μm.

Injection of the vectors without plasmin did not result in uniformmCherry expression in retinal bipolar cells across the entire retina(FIG. 4, control, top panels). Transduction efficiency was low in thecenter (A) and middle (B) retina, but high in the periphery.Co-injection of the AAV2 mCherry vector with increasing dosages ofplasmin (low, middle and high, bottom panels) resulted in increasedtransduction efficiency at each retinal region in a dose-dependentmanner.

The qualitative results from immunofluorescence images were verified bycell counting. Quantification of mCherry-expressing cells whenco-injected with or without plasmin showed that plasmin significantlyincreased the density of mCherry-expressing retinal cells. The increasein transduction efficiency with plasmin compared to control wasstatistically significant with all three doses of plasmin at the centerof the retina. Middle and high doses of plasmin resulted in astatistically significant increase in mCherry expression at themid-region and periphery of the retina. These results show that plasminenhances transduction efficiency throughout the retina, including theperipheral, middle, and center regions of the retina.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of enhancing the delivery of a therapeutic agent to an eyeof a subject comprising administering a plasmin or derivative thereofand the therapeutic agent to the eye.
 2. The method of claim 1, whereinthe plasmin or derivative thereof is a miniplasmin or a microplasmin(ocriplasmin).
 3. The method of claim 1, wherein the therapeutic agentis selected from a nucleic acid, a small molecule, an antibody, or apeptide.
 4. The method of claim 3, wherein the nucleic acid is a nucleicacid expression vector, a plasmid, or an siRNA.
 5. The method of claim4, wherein nucleic acid expression vector is a viral vector comprising atransgene.
 6. The method of claim 5, wherein the transgene is an opsin.7. The method of claim 6, wherein the opsin is selected from the groupconsisting of channelrhodopsin, halorhodopsin, melanopsin, pineal opsin,bacteriorhodopisin, and proteorhodopsin, or a functional variantthereof.
 8. The method of claim 7, wherein said transgene is operablylinked to a cell-specific promoter.
 9. The method of claim 8, whereinthe therapeutic agent is encapsulated in a nanoparticle, a polymer, or aliposome.
 10. The method of claim 9, wherein the therapeutic agent isselected from the group consisting of ranibizumab antibody FAB(Lucentis), VEGF Trap fusion molecule (VEGF Trap-Eye), macugen pegylatedpolypeptide (Pegaptanib), and bevacimzumab (Avastin).
 11. The method ofclaim 1, wherein the subject is suffering from an ocular disease ordisorder.
 12. The method of claim 1, wherein the plasmin or derivativethereof and the therapeutic agent are delivered concurrently orsequentially.
 13. The method of claim 1, wherein the therapeutic agentis delivered to a retinal cell.
 14. The method of claim 13, wherein theretinal cell is a retinal ganglion cell, a retinal bipolar cell, aretinal horizontal cell, an amacrine cell, a photoreceptor cell, Müllerglial cell, or a retinal pigment epithelial cell.
 15. The method ofclaim 1, wherein the administration is to the vitreous of the eye.
 16. Amethod of increasing light sensitivity or improving or restoring visionin a subject comprising administering a plasmin or derivative thereofand a viral vector that encodes an opsin to the vitreous of the eye. 17.The method of claim 16, wherein said opsin is selected from the groupconsisting of channelrhodopsin, halorhodopsin, melanopsin, pineal opsin,bacteriorhodopisin, and proteorhodopsin, or a functional variantthereof.
 18. The method of claim 16, wherein the subject has an oculardisease or disorder.